Cyclooxygenase-3 inhibitory withanolide compositions and method

ABSTRACT

Cyclooxygenase-2 enzyme inhibiting withanolides are described. In particular, compounds from  Withania somnifera  are the preferred source of the withanolides, although they can be from other plant sources. The COX-2 inhibition is selective over COX-1.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to the use of withanolides as selectivecyclooxygenase-2 (COX-2) inhibitors. The withanolides have little effecton COX-1.

(2) Description of Related Art

Withania somnifera (L) Dunal of solanaceae, is an erect evergreen shrubdistributed throughout the drier parts of India. W. somnifera, known asAswagandha, is well known for its use in Ayurvedic medicine. TheAswagandha root extract was reported as a folk remedy for adenopathy,arthritis, asthma, hypertension, inflammations, and rheumatism (Thakur,R. S., et al., Major medicinal plants of India; Ed.; Central Instituteof Medicinal and Aromatic Plants: Lucknow, India, 531 (1989)). Theleaves of W. somnifera were also used as a cure for several illnessesincluding tumors, inflammations, conjunctivitis and tuberculosis(Thakur, R. S., et al., Major medicinal plants of India; Ed.; CentralInstitute of Medicinal and Aromatic Plants: Lucknow, India, 531 (1989)).Currently, powdered roots or root extract of this plant are used as adietary supplement in the United States.

The major chemical constituents reported from W. somnifera are calledwithanolides. These compounds are structurally diverse steroidalcompounds with an ergosterol skeleton in which C-22 and C-26 areoxidized to form a δ-lactone; (Ray, A. B., et al., Prog. Chem. Org. Nat.Prod. 63, 1-106 (1994)). The chemical investigations of the roots andleaves of W. somnifera resulted in the isolation and characterization ofseveral withanolides (Matsuda, M., et al., Bioorg. Med. Chem. 9,1499-1507 (2001)). The fruits of this plant are tiny orange berries andreported to contain saturated and unsaturated fatty acids (Stoller E.W., et al., Lloydia, 37, 309-312 (1974); Monika, P., et al., Asian J.Chem. 6, 442-444 (1994); and Monika, P., et al., Wsci. Phys. Sci. 5,81-83 (1993)). However, leaves and fruits are not fully investigated forbiological activities. The withanolides are classified according totheir structural skeleton (Ray, A. B., et al., Prog. Chem. Org. Nat.Prod. 63, 1-106 (1994)) and the structural variation is responsible forthe wide array of pharmacological activities. Withanolides have beenstudied for their anti-inflammatory, antitumor, cytotoxic,immunomodulating activities and for the protection against CCl₄-inducedhepatotoxicity (Ray, A. B., et al., Prog. Chem. Org. Nat. Prod. 63,1-106 (1994); and Anjaneyulu, A. S. R., et al., Studies in NaturalProducts Chemistry: Structure and Chemistry (Part F); Ed.Atta-ur-Rahman, Vol. 20, 135-261 (1998)). They were also reported toinduce phase-II enzymes in animal models, which is considered to be oneof the mechanisms in cancer chemoprevention (Misico, R. I., et al., J.Nat. Prod. 65, 677-680 (2002); and Su, B. N., et al., Tetrahedron 58,3453-3466 (2002)).

Cyclooxygenase-1 (COX-1) and -2 (COX-2) enzymes are responsible for theconversion of arachidonic acid, a lipid present in the cell, toprostaglandins. Prostaglandins in turn cause inflammatory responses inthe body. Inhibition of COX-1 enzyme may result in the formation ofulcers in many human and hence the selective inhibition of COX-2 enzymeby compounds has a major advantage over non-selective nonsteroidalanti-inflammatory drug (NSAIDs) (Smith, W. L., et al., Anu. Rev.Biochem. 69:145-182 (2000)) sold over the counter (OTC). It is importantto note that over expression of COX-2 enzyme was observed not only ininflamed cells but also by various types of tumor cells (Patti, R., etal., Cancer Lett. 180:13-21 (2002); Ohno, R., et al., Cancer91:1876-1881 (2001); and Khuder, S. A., et al., British Journal ofCancer 84:1188-1192 (2001)). Hence, COX-2 inhibitors with little or noCOX-1 activity are of great interest for the chemoprevention of cancer.

OBJECTS

It is therefore an object of the present invention to providecompositions and a method for inhibiting COX-2 selectively. It isparticularly an object of the present invention to provide a method andcompositions which do not inhibit COX-1. These and other objects willbecome increasingly apparent by reference to the following descriptionand the drawings.

SUMMARY OF INVENTION

The present invention relates to a method for selectively inhibitingCOX-2 enzyme relative to COX-1 enzyme which comprises providing aneffective amount of a withanolide so as to produce the COX-2 inhibition.Preferably the withanolide is present in plant material of Withaniasomnifera. In particular, the present invention relates to a method forselectively inhibiting COX-2 enzyme relative to COX-1 enzyme whichcomprises providing an effective amount of an isolated and purifiedwithanolide or mixtures thereof present in Withania somnifera so as toproduce the COX-2 enzyme inhibition. The inhibition can be in vitro;however, preferably inhibition is in vivo in a mammal.

The method preferably uses a compound selected from the group consistingof physagulin D (1→6)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside;27-O-β-D-glucopyranosyl physagulin D; 27-O-β-D-glucopyranosylviscosalactone B; 4, 16-dihydroxy-5β, 6β-epoxyphysagulin D;4-(1-hydroxy-2,2-dimethylcyclo-propanone)-2, 3-dihydrowithaferin A;2,3-dihydrowithaferin A; viscosalactone B; sitoindoside IX; physagulinD; withanoside IV, withaferin A and mixtures thereof.

Further, the present invention relates to a composition which comprises:

a withanolide or mixtures thereof increased over an amount occurring innature; and

a pharmaceutically acceptable carrier, wherein the compositionselectively inhibits COX2 enzyme relative to COX1 enzyme.

Preferably the composition comprises:

an isolated and purified withanolide or mixtures thereof present inWithania somnifera; and

a pharmaceutically acceptable carrier, wherein the compositionselectively inhibits COX2 enzyme relative to COX1 enzyme. In thecomposition the compound is previously listed.

The present invention also relates to an isolated and purified compoundof the formula:

wherein R is Glc-(1→6)-Glc-(1→4)-Glc, where Glc is glucose, and whereinR′ is H.

The present invention also relates to an isolated and purified compoundof the formula:

wherein R is Glc and R′ is Glc, wherein Glc is glucose.

The present invention also relates to an isolated and purified compoundof the formula:

wherein R=O, R′=H, R″=H and R″′=Glc, wherein Glc is glucose.

The present invention relates to an isolated and purified compound ofthe formula:

wherein R=—OH, R′=Glc, R′=OH and R″′=H.

Finally, the present invention relates to a purified compound of theformula:

DESCRIPTION OF DRAWINGS

FIGS. 1 to 1D are chemical formulas showing the structures ofwithanolides 1 to 12.

FIGS. 2A to 2E are chemical formulas showing some of the significantHMBC (→) correlations observed in compounds 1, 2, 3, 4 and 5.

FIG. 3A is a graph showing inhibition of COX-1 and -2 enzymes bycommercial non-steroidal anti-inflammatory agents (NSAIDs). Aspirin,ibuprofen and naproxen were tested at 180, 2.1 and 2.5 μg/ml,respectively. Celebrex, Vioxx and Bextra were assayed at 1.67 μg/ml,respectively. DMSO solvent control did not inhibit COX enzymes. Data arerepresented as mean ±one standard deviation (n=2).

FIG. 3B is a graph showing COX-1 and -2 inhibitory activities ofwithanolides 1-12 at 100 μg/ml. DMSO solvent control did not inhibit COXenzymes. Vertical bars represent the standard deviation of each datapoint (n=2) . Withanolides 1-12 did not inhibit COX-1 enzyme even at 500μg/ml concentration.

FIGS. 4A and 4B are graphs showing dose dependent inhibition of COX-2enzyme by compounds 1-5 at 50, 100 and 250 μg/ml. Vertical barsrepresent the standard deviation of each data point (n=2).

FIG. 5 is a graph showing inhibition of lipid peroxidation at 20 min bycompounds 4, 7, 10, and 11 at 100 ppm and synthetic antioxidants BHA,BHT and TBHQ at 10 ppm concentration. DMSO, used as a solvent control,did not show activity. Similarly, withanolides 1-3, 5, 6, 8, 9, and 12did not show activity at 100 ppm concentration. Data are represented asmean ±one standard deviation (n=2).

DESCRIPTION OF PREFERRED EMBODIMENTS

It has been discovered that leaf extracts of W. somnifera possessexcellent selective COX-2 inhibitory activity. The isolation andcharacterization of several novel withanolides and a number of knownwithanolides from W. somnifera leaf extracts is disclosed. Theinhibitory effects of withanolides isolated from the leaves oncyclooxygenase enzymes and their antioxidant activities are alsodisclosed.

Four novel withanolide glycosides and a withanolide (herein all referredto as “withanolide”) have been isolated from the leaves of Withaniasomnifera. The structures of the novel compounds were elucidated asphysagulin D (1→6)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside (1),27-O-β-D-glucopyranosyl physagulin D (2), 27-O-β-D-glucopyranosylviscosalactone B (3), 4, 16-dihydroxy-5β, 6β-epoxyphysagulin D (4), and4-(1-hydroxy-2, 2-dimethylcyclo-propanone)-2,3-dihydrowithaferin A (5)on the basis of 1D-, 2D-NMR and MS spectral data. In addition, sevenknown withanolides withaferin A (6), 2,3-dihydrowithaferin A (7),viscosalactone B (8), 23, 24-dihydrowithaferin A (9), sitoindoside IX(10), physagulin D (11), and withanolide IV (12) were isolated. Thesewithanolides were assayed to determine their ability to inhibitcyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes and lipidperoxidation. The withanolides tested, except compound 9, showedselective COX-2 enzyme inhibition ranging from 9-40% at 100 μg/ml.Compounds 4, 10 and 11 also inhibited lipid peroxidation by 40, 44 and55%, respectively.

EXAMPLES 1 TO 12

The W. somnifera leaf extract purchased was identical to the methanolextract of fresh leaves. harvested from W. somnifera plants grown in thegreenhouses of Bioactive Natural Products and Phytoceuticals laboratoryat Michigan State University. The extract was purified by preparativeTLC and by HPLC to yield pure withanolides 1-12.

Compound 1 was isolated as an amorphous powder and its molecular formulawas determined as C₄₆H₇₃O₂₀ by HRFABMS as indicated by an [M+H]⁺ ion atm/z 945.4682 (calc. 945.4695). Compound 1 showed absorption bands in itsIR spectrum at 3406 and 1698 cm⁻¹, respectively, corresponding to an —OHand an α,β-unsaturated lactone moieties. Three anomeric protons doubletsat δ 4.22, 4.20 and 4.15 were correlated to three anomeric carbons at δ103.0, 103.9 and 104.8, respectively, in its HMQC spectrum and suggestedthat compound 1 contained a triglycosidic moiety. Apart from theglycosidic signals, compound 1 exhibited signals for 28 carbons. Thesignals at 80.1, 32.8, 160.4, 123.6, and 168.6 ppm were assigned to asix-membered ring α,β-unsaturated δ-lactone moiety in the molecule andthe olefinic carbons at δ 139.0 and 125.5 were assigned to C-5 and C-6.The DEPT spectrum of compound 1 showed the presence of three methinecarbons at δ 74.9, 73.6, 80.1 and a methylene carbon at δ 57.6 and wereindicative of C-1, C-3, C-22 and C-27 oxygenated carbons.

The singlets at δ 0.60, 0.90, and 1.90 and a doublet at 0.92 ppm in its¹H NMR were assigned to C-18, 19, 28 and 21, respectively. An olefinicproton at δ 5.50 was placed at C-6 as it showed correlations with thiscarbon at 125.5 ppm in its HMQC. The C-6′ and C-4″ of the sugar units incompound 1 appeared at 70 and 77.8 ppm, respectively. These carbonsnormally appear at around 62 and 71 ppm, respectively, in glucose thatare not conjugated. The attachment of one of the glucose units to C-3 ofthe aglycone was confirmed by the HMBC correlations (FIG. 2A) observedbetween H-1′ and δ 4.22 and C-3 at 73.6 ppm. Other HMBC correlationssignificant to glucose linkages in compound 1 (FIG. 2A) were H-1″ at δ4.20 to C-6′ at δ 70.0 and H-1″′ at δ 4.15 to C-4″ at 77.8 ppm,respectively. Acid hydrolysis of compound 1 gave only D-glucose and theaglycone. The ¹H-NMR spectral data of the aglycone was identical to thepublished spectral data of sominone (Atta-ur-Rahman; Jamal, S. A.,;Choudhary, M. I. Heterocycles 34:689-698 (1992)). Also, the ¹³C NMR dataof compound 1 was compared to sominone, obtained as a hydrolysis productof physagulin D (11) in our laboratory. Therefore, glucose linkages incompound 1 were established as[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(→4) β-D-glucopyranoside].The mass spectral fragments obtained at m/z 783, 621 and 459 forcompound 1 in its FABMS showed the successive loss of three glucoseunits which further confirmed the proposed structure of compound 1 asPhysagulin D (1→6)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside.

The IR spectrum of Compound 2, isolated as an amorphous powder, showedabsorption bands at 3407, 1696 cm⁻¹ were due to the hydroxyl and anα,β-unsaturated δ-lactone carbonyl functionalities. The mass spectrum of2 displayed an [M+H]⁺ ion at m/z 783.4168 (calc. 783.4188), which wasconsistent with the molecular formula as C₄₀H₆₃O₁₅. The ¹H NMR signalsappeared as singlets at δ 0.76, 1.01 and 2.11, respectively, wereassigned to three methyl groups in 2. It also showed one methyl doubletat δ 1.22, C-27 methylene protons as doublets, integrated for one protoneach, at δ 4.60 and 4.46, two oxymethine multiplets at δ 4.50 and 3.83and an olefinic proton at δ 5.49. ¹³C NMR of 2 exhibited signals for anα,β-unsaturated δ-lactone carbonyl at δ 168.6, two oxygenated methinesat δ 74.9 and 73.6 and olefinic carbons at δ 139.1 and 125.4. The NMRspectra of compound 2 were similar to that of 1, with a major differencedue to the absence of one of the sugars as indicated by the lack ofanomeric proton and carbon signals at δ 4.20 and 104.8, respectively.Acid hydrolysis of 2 gave D-glucose and sominone (Atta-ur-Rahman, etal., Heterocycles 34:689-698 (1992)), identified by comparing the TLCwith hydrolysis products of 1. Compound 2 gave a molecular ion at m/z783, which is 162 amu less than that of 1, suggested that its probablestructure was simonene diglucoside. One of the glucose units in compound2 was assigned at C-3 based on the HMBC correlations (FIG. 2B) observedbetween H-3 at δ 3.83 and C-1′ at δ 102.7. The second glucose unit incompound 2 was assigned at C-27 as this carbon at δ 63.5 was shifted todownfield by 5.9 ppm when compared to the chemical shift of similarcarbon in 1 at δ 57.6. The linkage in compound 2 was further supportedby its HMBC spectral correlations (FIG. 2B) observed between C-27 at δ63.5 and H-1″ at δ 4.31. The placement of sugar units at C-3 and C-27was further confirmed by comparison of ¹³C NMR data of A-ring andlactone ring carbons in 2 with that of physagulin D (11) andsitoindoside IX (10). Therefore, structure of 2 was concluded as27-O-β-D-glucopyranosyl physagulin D.

The HRFABMS of Compound 3, obtained as an amorphous powder, revealed an[M+Na]⁺ peak at m/z 673.3200 (calc. 673.3224) and corresponded to amolecular formula of C₃₄H₅₀O₁₂. The IR spectrum of compound 3 indicatedthe presence of hydroxyl, α,β-unsaturated δ-lactone and a six-memberedring ketone in the molecule as indicated by absorption bands at 3425,1700, 1652 cm⁻¹, respectively. The ¹H NMR spectrum of compound 3displayed signals for three oxygenated methine protons at δ 3.66, 3.33and 4.44, four methyls at δ 0.67, 0.98, 1.18, and 2.1 and oxymethyleneprotons at δ 4.59 and 4.45. It also showed a doublet for an anomericproton at δ 4.31 and a broad singlet at 3.15 ppm, which correlated tothe anomeric carbon at δ 102.7 and an epoxide carbon at δ 56.6 in itsHMQC, respectively. The ¹³C NMR spectrum showed signals due to carbonylcarbon of a keto group at δ 210.2; an epoxide moiety at δ 63.8 and 56.6and for a six-membered ring α,β-unsaturated lactone moiety at δ 78.9,29.6, 159.1, 122.5 and 167.4. The spectral data suggested that compound3 was closely related to viscosalactone B (8).

Hydrolysis of compound 3 gave viscosalactone B (8) and D-glucose asconfirmed by spectral studies. This indicated that compound 3 was aviscosalactone glucoside. Analyses of its HMBC spectrum (FIG. 2C)suggested that the glucose moiety was attached to C-27 as this carbon atδ 61.6 correlated with the anomeric proton at 4.31 ppm. Linkage of oneof the glucose units to C-27 was also supported by the downfield shiftof C-27 by 5.3 ppm as compared to its aglycone viscosalactone B (9),which appeared at δ 57.1. The spectral evidence S confirmed thestructure of compound 3 as 27-O-β-D-glucopyranosyl viscosalactone B (3).

Compound 4 was obtained as an inseparable mixture with compound 12 andthe ratio was about 2:1. The HRFABMS displayed an [M+H]⁺ ion at m/z669.3456 (calc. 669.3486) which corresponded to the molecular formulaC₃₄H₅₃O₁₃. The base peak at m/z 507, produced by the loss of 162 amufrom the molecular ion, indicated that compound 4 contained a monoglycoside. ¹H- and ¹³C NMR assignments for compound 4 were unambiguouslyassigned by supporting evidences from DEPT, HMQC and HMBC spectralstudies.

Apart from the sugar carbons, ¹³C NMR and DEPT spectra displayed signalsfor four methyl groups at δ 11.9, 13.6, 15.0 and 20.0, α,β-unsaturatedδ-lactone carbonyl at δ 168.5 and oxygenated methines at δ 79.0, 75.6,73.6, and 59.4. The molecular ion at m/z 669, 48 amu higher than that ofphysagulin D (m/z 621), indicated that compound 4 contained threeadditional oxygen functionalities in its structure. One such oxygenfunctionality, assigned as an epoxide at C-5 and C-6, resonated at δ65.5 and 59.4, respectively in its ¹³C NMR spectrum. The second andthird oxygen functionalities were determined as hydroxyl groups and wereplaced at C-4 and C-16 as confirmed by HMBC experiments (FIG. 2D).Comparison of ¹H and ¹³C NMR data of 4 with physagulin D indicated thatthe sugar moiety was glucose and it was linked to C-3 of the molecule.The linkage of glucose unit to C-3 was also substantiated by HMBCcorrelations (FIG. 2D) observed between C-3 at 6′ 73.6 and H-1′ at δ4.38. Therefore, the structure of 4 was confirmed as 4,17-dihydroxy-5β,6β-epoxyphysagulin D.

The IR spectrum of compound 5, isolated as an amorphous powder, showedabsorption bands for an —OH and a δ-lactone carbonyl group at 3434 cm⁻¹and 1704 cm⁻¹, respectively. The HRFABMS gave an [M+H]⁺ ion at m/z555.3335, which analyzed for C₃₃H₄₇O₇ (calc. 555.3323). The ¹³C NMR andDEPT spectra of compound 5 showed the presence of six methyl, ninemethylene, nine methine and nine quaternary carbons. The ¹H NMR spectrumof 5 showed signals for four methyl groups at δ 0.69, 0.98, 1.18, and2.07. Two doublets at δ 4.35 and 4.28 was assigned to two protons of amethylene at C-27. In addition, the proton signal appeared at δ 3.19 wasassigned to H-4. The ¹³C NMR spectrum of compound 5 was similar to thespectrum of dihydrowithaferin A (7) except that 5 had additional carbonsignals at δ 210.1, 52.1, 72.8, and 25.0. The corresponding protonsignals in 5 were at δ 1.35 (6H, s) and 3.70 (1H, s). These additionalcarbon signals accounted for a 2, 2-dimethylcyclopropanone moiety in 5,which was supported by both MS and DEPT spectral data. Analyses of theHMBC spectrum revealed that 2,2-dimethylcyclopropanone moiety in 5 waslinked via C-4 hydroxyl in dihydrowithaferin A (7) (FIG. 2E). Thefragments at m/z 472 [M+H-C₅H₇O]⁺ and 471 [M+H-C₅H₈O]⁺ in its FABMS alsosupported the proposed structure for compound 5.

The withanolides isolated from the leaves were evaluated for theircyclooxygenase (COX) enzyme inhibitory activity using prostaglandinendoperoxide synthase isozymes-1 (COX-1) and PGHS-2 (COX-2). Aspirin,ibuprofen, naproxen, Celebrex, and Bextra were used as positive controlsand they showed 61, 53, 79, 23 and 25% of COX-1; 7, 59, 95, 98 and 99%of COX-2 inhibition, respectively (FIG. 3A). Vioxx inhibited COX-2enzyme by 80% and had no COX-1 enzyme inhibition. The novel withanolides1-5 were tested at 50, 100 and 250 μg/ml and all other withanolidesisolated from the leaves were tested at 100 μg/ml concentration.Compounds 6, 7, 8, 10, 11, and 12 gave 39, 27, 35, 13, 14 and 23%,respectively, of COX-2 enzyme inhibition at 100 μg/ml (FIG. 3B). A dosedependent inhibition of COX-2 (FIG. 3) was observed for compounds 1-5and the activity varied considerably among withanolides atconcentrations tested. The COX-2 activity exhibited by compounds 1-5were 15, 9, 7, 5 and 15%, respectively, at 50 μg/ml (FIG. 4). It isimportant to note that the withanolides tested did not inhibit COX-1enzyme even at 500 μg/ml concentration. However, the activity remainedthe same for all compounds when the concentration was increased from 100to 250 μg/ml in COX-2 assays. The lack of increased COX-2 activity athigher concentrations is probably due to the solubility of thesewithanolides under assay conditions. The reduced COX-2 activity ofcompounds 3 and 10 as compared to 8 and 6 might be due to theglycosylation at C-27 in 3 and 10. Compound 9, which is lacking a doublebond between C-23 and C-24, showed neither COX-1 nor COX-2 activities.This indicated that the double bond in α,β-unsaturated δ-lactone moietyis critical for the COX-2 inhibitory activity.

The ability of withanolides to inhibit lipid peroxidation in a modelsystem was used to determine whether they could act as antioxidants. Theassay was conducted by using large unilamellar vesicles and peroxidationwas initiated by adding Fe²⁺. Except for compounds 4, 7, 10 and 11,other withanolides tested did not inhibit the lipid peroxidation (FIG.5). The monoglycosides 4, 7, 10, and 11 inhibited 40, 5, 44 and 55%,respectively, of lipid peroxidation in our assay system.

In vitro results on the COX-2 enzyme inhibitory activities ofwithanolides provided some scientific support for the use of W.somnifera leaf preparation as a folk remedy for the treatment ofinflammation (Thakur, R. S., et al., Major medicinal plants of India;Ed.; Central Institute of Medicinal and Aromatic Plants: Lucknow, India,p. 531 (1989)). This invention also represents the first report of theCOX-2 enzyme inhibitory activity for this group of compounds. Overexpression of COX-2 enzyme was observed in tumor cells and henceselective COX-2 inhibitors can prevent tumor progression. Anecdotalreports indicate that the withanolides exhibit anticancer activity(Thakur, R. S., et al., Major medicinal plants of India; Ed.; CentralInstitute of Medicinal and Aromatic Plants: Lucknow, India, p. 531(1989)). Therefore, these compounds can be useful as templates for thedevelopment of therapeutics for cancer chemoprevention. Since both rootsand leaves of W. somnifera contain similar withanolides, consumption ofW. somnifera root powder or leaf extract as a dietary supplement candecrease the inflammatory pain, the risk of cancer formation andprogression of tumors at levels which suppress the COX-2 enzyme.

Experimental Procedures

General. ¹H NMR spectra were recorded on a 500 MHz VRX spectrometer. ¹³CNMR spectra were obtained at 125 MHz. Chemical shifts were recorded ineither CDCl₃ or CD₃OD. HMBC was optimized for J=8.0 Hz. The silica gelused for MPLC was Merck Silica gel 60 (35-70 μm particle size). HRFABand EAB mass spectra were acquired on JEOL HX-110 double focusing massspectrometer operating in the positive mode. Preparative HPLC wasperformed on a recycling preparative HPLC (Japan Analytical Industry Co.model LC-20) with tandem C₁₈ column (JAIGEL, 10 μm, 20×250 mm) at theflow rate of 3 ml/min. All organic solvents and standards used were ACSreagent grade. Yields of the withanolides are expressed in percentagedry weight of the leaves.

Plant Material. The extract (H-341) was purchased from PhytoMycoResearch Corporation, Greenville, N.C. and was prepared as follows: Theshade dried and ground leaves of W. somnifera were extractedsequentially with a mixture of dichloromethane and methanol (1:1, v/v),methanol and water to obtain three fractions. All fractions were thenpooled, filtered and dried under reduced pressure. The resulting extractwas stored at −20° C. until use.

Isolation of withanolides. The combined crude extract from PhytoMycoResearch Corporation (4 g) was stirred with n-hexane (500 mL) andfiltered. The hexane insoluble portion (3.2 g) was chrematographed onMPLC using CHCl₃ and MeOH (v/v) under gradient condition. The fractionscollected were I (600 mg CHCl₃:MeOH, 9:1), II (500 mg, CHCl₃:MeOH, 8:2),III (1.2 g, CHCl₃:MeOH, 7:3), and IV (200 mg, CHCl₃:MeOH, 1:1). RepeatedMPLC of fraction I using hexane-EtOAc (1:1, v/v) yielded pure compounds6 (120 mg, 0.078%), 7 (50 mg, 0.032%) and a fraction 1 (4.0 mg). Thisfraction was further purified by PTLC (hexane-EtOAc, 6:4, v/v) to yieldthe pure compound 9 (2.1 mg, 0.0014%). Purification of fraction II bypreparative HPLC using MeOH—H₂O (1:1, v/v) afforded compound 10 (20 mg,0.013%) at 19.5 min and fraction 2 (50 mg) at 24.2-28.1 min. It wasfurther purified on HPLC by using MeOH—H₂O (4:6, v/v) and gave compound11 (40 mg, 0.026%) at 32.96 min. Similarly, fraction III was purified byPrep. HPLC (MeOH—H₂O, 6:4, v/v) and yield pure compound 12 (15 mg,0.0097%) at 112 min and fractions 3 (950 mg) at 20-45 min and 4 (16 mg)at 140-160 min. Fraction 3 was further purified by HPLC (MeOH—H₂O, 6:4,v/v) to afford compounds 8 (200 mg, 0.13%) and 5 (10 mg, 0.0065%) at66.8 and 78.6 min, respectively, and 700 mg of sucrose at 132 min.Fraction 4 was subjected to HPLC (MeOH—H₂O, 7:3, v/v) yielded compound 2(12 mg, 0.0078%) at 81 min. The fraction IV was purified by HPLC(MeOH—H₂O, 7:3) and collected fractions 5 (13 mg) at 20-30 min andfraction 6 (30 mg) at 30.1-66 min. The fraction 6 was purified by HPLC(MeOH—H₂O, 7:3, v/v) to yield compound 3 (8.5 mg, 0.0055%) at 57.9 min.Fraction 5 was further purified by HPLC using MeOH—H₂O (6:4, v/v) andafforded pure compound 1 (10.5 mg, 0.0068%) at 59.3 min and a mixture ofcompounds 4 and 12 at 96.4 min (5.0 mg, 0.0032%).

Compound 1. Colorless, amorphous powder; IR ν_(max) (KBr) 3406, 1698,1650, 1383, 1076, 1042 cm⁻¹; ¹H NMR (CD₃OD) δ 0.60 (3H, s, Me-18), 0.90(3H, s, Me-19), 0.92 (3H, d, J=7.0 Hz, Me-21), 1.90 (3H, s, Me-28), 3.96(1H, m, H-3), 4.15 (1H, d, J=8.0 Hz, H-1″′), 4.20 (1H, d, J=8.0 Hz,H-1″), 4.22 (1H, d, J=8.0 Hz, H-1′), 4.30 (1H, t, J=2.3 Hz, H-1), 4.35(2H, br s, H-27), 4.48 (1H, dt, J=13.0, 3.4 Hz, H-22), 5.50 (1H, br. D,J=5.5 Hz, H-6); ¹³C NMR data (Table 1); FABMS m/z 945 (M+H⁺), 783(M+H⁺-glucose), 621 (M+H⁺-2×glucose), 459 (aglycone); HRFABMS m/z945.4682 (M+H⁺; calcd for C₄₆H₇₃O₂₀, 945.4695). TABLE 1 ¹³C NMR chemicalshifts of withanolides 1-5^(a) Carbon number 1 2 3 4 5  1 74.9 d 74.9 d210.2 s 75.0 d 210.6 s  2 37.7 t 37.7 t 42.6 t 37.8 t 52.2 t  3 73.6 d73.6 d 70.4 d 73.6 d 40.5 t  4 39.2 t 39.1 t 73.8 d 75.6 d 72.8 d  5139.0 s 139.1 s 63.8 s 65.5 s 64.2 s  6 125.5 d 125.4 d 56.6 d 59.4 d57.1 d  7 30.7 t 30.8 t 29.8 t 30.7 t 30.0 t  8 33.2 d 33.2 d 31.0 d33.2 d 31.5 d  9 42.7 d 42.7 d 42.5 d 42.7 d 43.0 d 10 42.5 s 42.6 s49.1 s 53.0 s 49.5 s 11 21.3 t 21.3 t 20.7 t 22.3 t 21.1 t 12 40.4 t40.3 t 27.1 t 28.2 t 27.5 t 13 43.9 s 43.9 s 42.6 s 44.1 s 42.9 s 1457.2 d 57.6 s 56.1 d 51.7 d 55.7 d 15 25.5 t 25.5 t 24.2 t 39.2 t 24.6 t16 28.3 t 28.3 t 39.1 t 79.1 d 39.4 t 17 53.2 d 53.2 d 51.8 d 57.2 d50.1 d 18 12.1 q 12.1 q 10.7 q 11.9 q 11.1 q 19 20.0 q 19.9 q 13.6 q15.0 q 14.0 q 20 40.8 d 40.8 d 39.0 d 40.9 d 39.6 d 21 13.7 q 13.7 q12.5 q 13.6 q 12.9 q 22 80.1 d 80.2 d 78.9 d 80.2 d 79.4 d 23 32.8 t32.8 t 29.6 t 32.5 t 30.2 t 24 160.4 s 160.2 s 159.1 s 157.8 s 157.0 s25 123.6 s 123.6 s 122.5 s 125.4 s 125.6 s 26 168.6 s 168.6 s 167.4 s168.5 s 167.7 s 27 57.6 t 63.5 t 61.6 t 57.6 t 56.6 t 28 20.7 q 20.7 q19.5 q 20.0 q 19.5 q  1′ 103.0 d 102.7 d 102.7 d 103.0 d 72.8 d  2′ 75.1d 75.1 d 75.6 d 75.6 d 52.1 s  3′ 78.0 d 78.1 d 76.9 d 78.0 d 210.1 s 4′ 71.5 d 71.7 d 72.4 d 71.7 d 25.4 q (4′& 5′- Me)  5′ 77.9 d 78.0 d76.8 d 77.9 d  6′ 70.0 t 62.9 t 62.4 t 62.7 t  1′′ 103.9 d 103.9 d  2′′75.0 d 75.0 d  3′′ 78.0 d 78.0 d  4′′ 77.8 d 71.6 d  5′′ 77.9 d 77.9 d 6′′ 62.7 t 62.8 t  1′′′ 104.8 d  2′′′ 75.0 d  3′′′ 78.0 d  4′′′ 71.5 d 5′′′ 77.9 d  6′′′ 63.5 t^(a)Data recorded in CD₃OD at 125 MHz at 25° C. Multiplicities weredetermined by DEPT experiments and confirmed by analysis of HMQCspectra.

Compound 2. Colorless, amorphous powder; IR ν_(max) (KBr) 3407, 1696,1652, 1398, 1040 cm⁻¹; ¹H NMR (CD₃OD) δ 0.76 (3H, s, Me-18), 1.01 (3H,s, Me-19), 1.22 (3H, d, J=7.0 Hz, Me-21), 2.11 (3H, s, Me-28), 3.83 (1H,m, H-3), 4.31 (1H, d, J=8.0 Hz, H-1″), 4.36 (1H, d, J=8.0 Hz, H-1′),4.46 (1H, d, J=11.5 Hz, H-27b), 4.48 (1H, dt, J=13.0, 3.4 Hz, H-22),4.50 (1H, t, J=2.3 Hz, H-1), 4.60 (1H, d, J=11.5 Hz, H-27a), 5.49 (1H,br. d, J=5.5 Hz, H-6); ¹³C NMR data (Table 1); FABMS m/z 783 (M+H)⁺);HRFABMS m/z 783.4168 (M+H⁺; calcd. For C₄₀H₆₃O₁₅, 783.4188).

Compound 3. Colorless, amorphous powder; IR ν_(max) (KBr) 3425, 1700,1652, 1400, 1259, 1058 cm⁻¹; ¹H NMR (CD₃OD) δ 0.67 (3H, s, Me-18), 0.98(3H, d, J=7.0 Hz, Me-21), 1.18 (3H, s, Me-19), 2.10 (3H, s, Me-28), 3.15(1H, br s, H-6), 3.33 (1H, d, J=8.0 Hz, H-4), 3.66 (dd, 1H, J=11.0, 3.0Hz, H-3), 4.31 (1H, d, J=7.5 Hz, H-1′), 4.44 (1H, dt, J=13.5, 3.5 Hz,H-22), 4.45 (1H, d, J=11.5 Hz, H-27b), 4.59 (1H, d, J=11.5 Hz, H-27a);¹³C NMR data (Table 1); FABMS m/z 673 ([M+Na]⁺). HRFABMS m/z 673.3200([M+Na]⁺; calcd. for C₃₄H₅₀O₁₂, 673.3224).

Compound 4. Colorless, amorphous powder; IR ν_(max) (KBr) 3424, 1697,1652, 1398, 1384, 1076, 1042 cm⁻¹; ¹H NMR (CD₃OD) δ 0.69 (3H, s, Me-18),1.0 (3H, d, J=7.0 Hz, Me-21), 1.22 (3H, s, Me-19), 2.08 (3H, s, Me-28),3.14 (1H, br s, H-6), 3.41 (br t, J=7.5 Hz, H-16), 3.43 (1H, d, J=8.0Hz, H-4), 3.60 (1H, m, H-3), 4.19 (1H, br s, H-27), 4.38 (1H, d, J=7.5Hz, H-1′), 4.30 (1H, dt, J=13.5, 3.4 Hz, H-22), 4.46 (1H, t, J=2.3 Hz,H-1); ¹³C NMR data (Table 1); FABMS m/z 669 (M+H⁺), 507 (aglycone);HRFABMS m/z 669.3456 (M+H⁺; calcd. For C₃₄H₅₃O₁₃, 669.3486).

Compound 5. Colorless, amorphous powder; IR ν_(max) (KBr) 3434, 1704,1634, 1397, 1382, 1256, 1236, 1058, 999 cm⁻¹; ¹H NMR (CD₃OD) δ 0.69 (3H,s, Me-18), 0.98 (3H, d, J=6.5 Hz, Me-21), 1.18 (3H, s, Me-19), 1.35 (6H,s, Me-4′ & 5′), 2.07 (3H, s, Me-28), 3.15 (1H, br s, H-6), 3.19 (1H, t,J=2.3 Hz, H-4), 3.70 (1H, s, Hz, H-1′), 4.28 (1H, d, J=12.0 Hz, H-27),4.35 (1H, d, J=12.0 Hz, H-27), 4.42 (1H, dt, J=13.0, 3.0 Hz, H-22); ¹³CNMR data (Table 1); FABMS m/z, 555 (M+H⁺), 472 [M+H—C₅H₇O]⁺, 471[M+H—C₅H₈O]⁺; HRFABMS m/z 555.3335 (M+H⁺; calcd. For C₃₄H₅₀O₁₂,555.3323).

Acid hydrolysis. Compounds 1-5 each 1 mg and 11 (5 mg) were dissolvedseparately in 6% HCl and heated under reflux for 3 hours. This solutionwas neutralized with 1N NaOH and extracted with EtOAc. The EtOAc extractwas concentrated and the aglycone was characterized by spectralexperiments. The aqueous solution was concentrated and analyzed forsugars. It was determined that only D-glucose was present in the aqueousportion.

Compounds 6-12. The structures of compounds 6-12 were derived aswithaferin A (6) (Anjaneyulu, A. S. R., et al., Indian J. Chem. Sect. B.36:161-165 (1997)); 2,3-dihydrowithaferin A (7) (Anjaneyulu, A. S. R.,et al., Indian J. Chem. Sect. B. 36:161-165 (1997)); viscalactone B (8)(Pelletier, S. W., et al., Heterocycles 15:317-320 (1981)); 23,24-dihydrowithaferin A (9) (Kirson, I., et al., Tetrahedron 26:2209-2219(1970)); sitoindoside IX (10) (Ghosal, B., et al., Ind. J. Nat. Prod.4:12-13 (1988)); physagulin D (11) (Shingu, K., et al., Chem. Pharm.Bull. 40:2088-2091 (1992)); and withanolide IV (12) (Matsuda, M., etal., Bioorg. Med. Chem. 9:1499-1507 and references cited therein (2001))by detailed ¹H and ¹³C NMR spectral experiments. The spectral data ofthese compounds were identical to their respective published spectraldata.

CD analysis. CD spectra for compounds 1-5 were recorded on a JASCO,model J-710, CD-ORD spectrometer in MeOH under the following conditions:scan mode (wave length), band width (0.5 nm), sensitivity (50 m deg),response (1 sec), wave length range (200-400 nm), step resolution (1nm), scan speed (100 nm min⁻¹), and accumulation (1). The CD maximum orminimum (Δε) observed for compounds 1-5 were: 1 (c 0.001, MeOH) Δε+73.7(257); 2 (c 0.0005, MeOH) Δε+15.4 (262.5); 3 (c 0.001, MeOH) Δε+4.7(261); 4 (c 0.0005, MeOH) Δε+61.6 (260) and 5 (c 0.001, MeOH) Δε+9.8(260).

EXAMPLE 13

Cyclooxygenase enzyme inhibitory assay. COX-1 enzyme was prepared fromram seminal vesicles and COX-2 enzyme was isolated from insect cellscloned with human PGHS-2 enzyme. The inhibitory effects of testcompounds on COX-1 and -2 were measured by monitoring the initial rateof O₂ uptake using an oxygen electrode (Instech Laboratories, PlymouthMeeting, Pa.) attached to a biological oxygen monitor (Yellow SpringInstrument, Inc., Yellow Spring, Ohio) at 37° C. The enzymes werediluted (1:1) with Tris buffer (pH 7, 10-15 μl) and the test compounds(100 μg/ml, 10 μl) dissolved in DMSO were added to the assay mixturecomposed of 3 ml of 0.1 M Tris HCl, pH 7, 1 mmol phenol and 85 μg ofhemoglobin. The mixture was incubated for 2-3 min and reaction wasinitiated by the addition of arachidonic acid (10 μl of 1.64 μMsolution). The instantaneous inhibition was measured by using Quick LogData acquisition and control computer software (Strawberry Tree Inc.,Sunnyvale, Calif., USA). Positive controls aspirin, ibuprofen, naproxenwere tested at 180, 2.1 and 2.5 μg/ml, respectively, and Celebrex, Vioxxand Bextra were tested at 1.67 μg/ml. DMSO was used as solvent control(Wang, H., et al., J. Nat. Prod. 62:294-296 (1999)). The results areshown in FIGS. 3A and 3B. FIG. 4 shows the results with withanolides 1to 5.

EXAMPLE 14

Antioxidant activity. Large Unilamellar Vesicles (Liposome suspension)were prepared according to the published procedure (Ramsewak, R. S., etal., Phytomedicine 7:303-308 (2000). The final assay volume was 2 ml,consisting of 100 μl HEPES buffer (50 mM HEPES and 50 mM TRIS), 200 μl1M NaCl, 1.64 ml of N₂-sparged water, 20 μl of test sample or DMSO and20 μl of liposome suspension. The peroxidation was initiated by theaddition of 20 μl of FeCl₂.4H₂O (0.5 mM). The fluorescence was monitoredat 0, 1, 3 and every 3 min up to 21 min using a Turner Model 450 DigitalFluorometer. The decrease of relative fluorescence intensity over thetime indicated the rate of peroxidation. The percentage of inhibitionwas calculated with respect to DMSO control. All compounds were testedat 100 μg/ml and the positive controls BRA, BHT and TBHQ were tested at10 μM. The results are shown in FIG. 5.

Pharmaceutical Compositions

In pharmaceutical compositions, the withanolide is inhibitory at adosage of 1 to 1,000 micrograms per milliliter or gram. In a preferredembodiment, one or more of the withanolides for treating a patient areprovided to the patient at an inhibitory dose in a pharmaceuticallyacceptable carrier. As such, the withanolides are processed withpharmaceutical carrier substances by methods well known in the art suchas by means of conventional mixing, granulating, coating, suspending andencapsulating methods, into the customary preparations for oral orrectal administration. Thus, withanolide preparations for oralapplication can be obtained by combining one or more of theanthraquinones with solid pharmaceutical carriers; optionallygranulating the resulting mixture; and processing the mixture orgranulate, if desired and/or optionally after the addition of suitableauxiliaries, into the form of tablets or dragee cores.

Suitable pharmaceutical carriers for solid preparations are, inparticular, fillers such as sugar, for example, lactose, saccharose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example, tricalcium phosphate or calcium hydrogen phosphate; alsobinding agents, such as starch paste, with the use, for example, ofmaize, wheat, rice or potato starch, gelatine, tragacanth, methylcellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl celluloseand/or polyvinylpyrrolidone, esters of polyacrylates orpolymethacrylates with partially free functional groups; and/or, ifrequired, effervescent agents, such as the above-mentioned starches,also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, oralginic acid or a salt thereof, such as sodium alginate. Auxiliaries areprimarily flow-regulating agents and lubricating agents, for example,silicic acid, talcum, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate. Dragee cores are provided with suitablecoatings, optionally resistant to gastric juices, whereby there areused, inter alia, concentrated sugar solutions optionally containing gumarabic, talcum, polyvinylpyrrolidone, and/or titanium dioxide, lacquersolutions in aqueous solvents or, for producing coatings resistant tostomach juices, solutions of esters of polyacrylates orpolymethacrylates having partially free functional groups, or ofsuitable cellulose preparations such as acetylcellulose phthalate orhydroxypropyl-methylcellulose phthalate, with or without suitablesofteners such as phthalic acid ester or triacetin. Dyestuffs orpigments may be added to the tablets or dragee coatings, for example foridentification or marking of the various doses of active ingredient.

Withanolide preparations comprising one or more of the anthraquinoneswhich can be administered orally further include hard gelatine capsules,as well as hard or soft closed capsules made from gelatine and, ifrequired, a softener such as glycerin or sorbitol. The hard gelatinecapsules can contain one or more of the withanolides in the form of agranulate, for example in admixture with fillers such as maize starch,optionally granulated wheat starch, binders or lubricants such astalcum, magnesium stearate or colloidal silicic acid, and optionallystabilizers. In closed capsules, the one or more of the withanolides isin the form of a powder or granulate; or it is preferably present in theform of a suspension in suitable solvent, whereby for stabilizing thesuspensions there can be added, for example, glycerin monostearate.

Other withanolide preparations to be administered orally are, forexample, aqueous suspensions prepared in the usual manner, whichsuspensions contain the one or more of the anthraquinones in thesuspended form and at a concentration rendering a single dosesufficient. The aqueous suspensions either contain at most small amountsof stabilizers and/or flavoring substances, for example, sweeteningagents such as saccharin-sodium, or as syrups contain a certain amountof sugar and/or sorbitol or similar substances. Also suitable are, forexample, concentrates or concentrated suspensions for the preparation ofshakes. Such concentrates can also be packed in single-dose amounts.

Suitable withanolide preparations for rectal administration are, forexample, suppositories consisting of a mixture of one or more of thewithanolides with a suppository foundation substance. Such substancesare, in particular, natural or synthetic triglyceride mixtures. Alsosuitable are gelatine rectal capsules consisting of a suspension of theone or more of the withanolides in a foundation substance. Suitablefoundation substances are, for example, liquid triglycerides, of higheror, in particular, medium saturated fatty acids.

Likewise of particular interest are preparations containing the finelyground one or more of the withanolides, preferably that having a medianof particle size of 5 μm or less, in admixture with a starch, especiallywith maize starch or wheat starch, also, for example, with potato starchor rice starch. They are produced preferably by means of a brief mixingin a high-speed mixer having a propeller-like, sharp-edged stirringdevice, for example with a mixing time of between 3 and 10 minutes, andin the case of larger amounts of constituents with cooling if necessary.In this mixing process, the particles of the one or more of thewithanolides are uniformly deposited, with a continuing reduction of thesize of some particles, onto the starch particles. The mixturesmentioned can be processed with the customary, for example, theaforementioned, auxiliaries into the form of solid dosage units; i.e.,pressed for example into the form of tablets or dragees or filled intocapsules. They can however also be used directly, or after the additionof auxiliaries, for example, pharmaceutically acceptable wetting agentsand distributing agents, such as esters of polyoxyethylene sorbitanswith higher fatty acids or sodium lauryl sulphate, and/or flavoringsubstances, as concentrates for the preparation of aqueous suspensions,for example, with about 5- to 20-fold amount of water. Instead ofcombining the withanolide/starch mixture with a surface-active substanceor with other auxiliaries, these substances may also be added to thewater used to prepare the suspension. The concentrates for producingsuspensions, consisting of the one or more of the withanolide/starchmixtures and optionally auxiliaries, can be packed in single-doseamounts, if required in an airtight and moisture-proof manner.

In addition, the one or more withanolides can be administered to apatient intraperitoneally, intranasally, subcutaneously, orintravenously. In general, for intraperitoneal, intranasal,subcutaneous, or intravenous administration, one or more of thewithanolides are provided by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives. Preferably, the one or morewithanolides are provided in a composition acceptable forintraperitoneal, subcutaneous, or intravenous use in warm-bloodedanimals or humans. For example, such compositions can comprise aphysiologically acceptable solution such as a buffered phosphate saltsolution as a carrier for the one or more anthraquinones. Preferably,the solution is at a physiological pH. In particular embodiments, thecomposition is injected directly into the patient perfused through thetumor by intravenous administration.

Preparations according to the present invention comprise one or more ofthe withanolides at a concentration suitable for administration towarm-blooded animals or humans which concentration is, depending on themode of administration, between about 0.3% and 95%, preferably betweenabout 2.5% and 90%. In the case of suspensions, the concentration isusually not higher than 30%, preferably about 2.5%; and conversely inthe case of tablets, dragees and capsules with the one or more of theanthraquinones, the concentration is preferably not lower than about0.3%, in order to ensure an easy ingestion of the required doses of theone or more withanolides. The treatment of patients with thepreparations comprising one or more of the withanolides is carried outpreferably by one or more administrations of a dose of the one or morewithanolide which over time is sufficient to substantially inhibitCOX-2. If required, the doses can be administered daily or divided intoseveral partial doses which are administered at intervals of severalhours. In particular cases, the preparations can be used in conjunctionwith or following one or more other therapies such as radiation orchemotherapy. The administered dose of the one or more withanolides isdependent both on the patient (species of warm-blooded animal or human)to be treated, the general condition of the patient to be treated, andon the type of disease to be treated.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

1-10. (canceled)
 11. An isolated and purified compound of the formula:

wherein R is Glc and R′ is Glc, wherein Glc is glucose. 12-16.(canceled)